PROJECT SUMMARY Basal cells (BCs) of mammalian airways are resident stem cells of the pseudostratified epithelium of the airways and understanding how they are regulated and maintained will be important for understanding their aberrant growth in disease, such as COPD, cystic fibrosis and cancer, as well as for developing novel methods for harnessing BCs for the purposes of regenerative medicine. The Hippo signaling pathway has emerged as an important regulator of BC growth. Hippo signaling controls the activity of the transcriptional regulators YAP and TAZ (YAP/TAZ) by restricting nuclear YAP/TAZ localization in mature differentiated airway cells. Nuclear YAP activity has been shown to promote BC growth in the airways and has been shown to play important roles in a variety of stem cell populations in other tissues and organs. YAP is implicated in maintaining the airway BC fate through direct association with the basal cell specific transcription factor, p63. Airway basal stem cells play important roles lung injury repair, but the signals that regulate their growth and self-renewal are poorly understood. Our studies, as well as others, indicate that the nuclear activity of the transcriptional regulator YAP plays important roles in airway basal cell growth and in this proposal, we aim to understand the roles and regulation of YAP activity in this context. In this proposal we aim to use genetic mouse models which contain conditional knockouts of Hippo pathway regulating kinases, LATS1 and LATS2, in airway basal cells to understand how Hippo inactivity and subsequent YAP nuclear activity influences BC maintenance through its interaction with p63. Our preliminary data suggest that LATS1/2 deletion in Krt5 positive basal cells leads to basal cell hyperplasia and the formation of distinct populations of basal cells. Those adjacent to the basement membrane have the highest levels of nuclear p63, whereby this expression is reduced or lost in the Krt5+ cells that are more luminal in the hyperplastic lesion. Our studies further suggest that YAP activity in BCs is influenced by signals, such as FGF10, that come from stromal cells in the basement membrane, and we hypothesize that these signals influence YAP interactions with p63 to guide a BC self-renewal transcriptional program. We aim to unveil novel biochemical pathways that describe how signals from the basement membrane are transduced through BC receptor tyrosine kinases to increase YAP-p63 functional activity and BC self-renewal and growth. Specifically, we propose: 1) to determine if LATS1/2-depletion and subsequent YAP activation in airway basal cells influences BC maintenance through YAP-p63 interactions; 2) how signals from the basement membrane may promote these YAP/p63 interactions; and 3) if YAP-p63 complexes differentially regulate gene expression to promote BC fate. Ultimately this study will offer molecular insight into how BC self-renewal and expansion is regulated, which may offer directions for preventing BC expansion in disease or guide new methods to expand BCs ex vivo for regenerative therapy.